The discovery of a high positive DC potential in the scala media (EP) and of large Na+ and K+ gradients between cochlear endolymph and perilymph nearly forty years ago has led to extensive research aimed at determining how these gradients are formed and the role they play in auditory function. As a result, the influence of the EP and of the high K+ level in endolymph on the transduction of mechanical to electrical signals by hair cells is now well understood. However, little is yet known about the specific cellular and molecular mechanisms that produce the unique ion and electrical gradients in the inner ear. Such knowledge is essential because imbalances in ionic or electrical gradients are involved in hearing losses associated with metabolic disorders, ischemic events, Meniere's disease and aging. It also is probable that alterations in cellular ion homeostasis promote the damage to hair cells induced by noise and ototoxic drugs. The research proposed here aims at defining basic cellular and molecular mechanisms responsible for establishing and maintaining the inner ear's unique electrochemical gradients. As a first Specific Aim, experiments are designed to identify and clone nucleotide sequences specific to inner ear ion transport proteins and their isoforms. Once identified, riboprobes will be generated and antibodies will be raised against synthetic peptides derived from these sequences. As a second Specific Aim, these newly generated riboprobes and antibodies along with others obtained from collaborative and commercial sources will be employed to determine by in situ hybridization and immunohistochemistry the precise cellular and subcellular location of ion transport proteins in the inner ear of adult animals. Specific Aim 3 seeks to explore the physiological significance of these transport mediators by examining their expression in relation to the establishment of ion gradients and of the EP during onset and maturation of hearing in neonatal animals.